1. Field of the Invention
The present invention relates to a method for estimating the number of nuclei of a preselected isotope in a molecular species from an NMR spectrum, to a use of said method and to a computer program product for performing said method.
2. Description of the Related Art
Nuclear magnetic resonance spectroscopy is a well-known technique that is extensively applied for qualitative and quantitative analysis of a large variety of samples. The technique generally involves recording a nuclear magnetic resonance spectrum, henceforth called NMR spectrum, under conditions that are selective for a preselected nuclear isotope with non-zero spin angular momentum, such as 1H, 13C or many others. In general, an NMR spectrum obtained from a sample containing a molecular species comprises a plurality of signal peaks resulting from the nuclei of the preselected isotope. Each signal peak corresponds to a particular resonance frequency that is attributable to one or several nuclei experiencing a particular local magnetic field as a consequence of the particular molecular environment. Accordingly, the resonance frequency at which an NMR signal peak is observed, usually expressed in terms of the so-called chemical shift given in parts per million (ppm) with respect to a reference signal, is an indication of the molecular location of the nucleus or nuclei giving rise to the signal peak.
An important advantage of NMR spectroscopy as compared to other analytical techniques lies in the fact that under certain well-known conditions the integral of a signal peak is directly proportional to the number of resonating nuclei (see e.g. R. R. Ernst, G. Bodenhausen and A. Wokaun, Principles of Nuclear Magnetic Resonance in One and Two Dimensions, Oxford Science Publication, 1988, 91–157). Therefore, the integrals of the various signal peaks in an NMR spectrum reflect the number of nuclei contributing to each signal peak. This fact is routinely used as a guide for manual, i.e. non-automated interpretation of NMR spectra of unidentified molecular species, i.e. for qualitative analysis.
Moreover, NMR spectroscopy may also be used for quantitative analysis. Because of the above mentioned proportionality between integrals of signal peaks and numbers of resonating nuclei, the absolute integral of an NMR signal peak is directly related to the number of molecules containing the resonating nuclei that are present in the detection volume of the NMR spectrometer. However, the absolute integral of an NMR signal peak will generally depend on a host of experimental conditions. Quantitative analysis by means of NMR spectroscopy thus requires comparison of the measured signal integrals with an integral derived from a calibration signal.
Several methods for generating a suitable calibration signal are known in the art, as described e.g. in French Patent Application FR 2 735 865 A1. In particular, FR 2 735 865 A1 discloses a device and a method for quantitative analysis by NMR spectroscopy involving a synthetic NMR calibration signal produced by an electronic device. This technique has become known under the acronym “ERETIC” (standing for “Electronic REference To access In vivo Concentrations”) as described in: L. Barantin, A. Le Pape and S. Akoka, A new method for absolute quantitation of MRS metabolites. Magn. Res. Methods Vol. 38 (1997) 179–182.
A fundamental problem in both qualitative and quantitative applications of NMR spectroscopy is caused by the fact that any NMR spectrum obtained in practice will deviate to some degree from the theoretical expectation. This discrepancy is mainly due to inevitable noise contributions, but also to other experimental influences such as field inhomogeneities, drifts and further effects. Moreover, solvent effects and exchange reactions, e.g. exchange of hydrogen ions when carrying out 1H-NMR spectroscopy may lead to inaccuracies in the determination of integrals. As a consequence, the integral of a signal peak as compared to others of the same molecular species will generally differ somewhat from the theoretical expectation. A further problem is caused by the so-called chemical shift degeneracy, which refers to a situation where nuclei at different locations within a molecular species have essentially identical chemical shifts and thus give rise to overlapping NMR signal peaks. These facts give rise to the following difficulties in qualitative and quantitative analysis by NMR-spectroscopy.
In qualitative analysis, the relative integrals of all the signal peaks in a particular NMR spectrum of a molecular species should be resealed, i.e. the integral of each signal peak should be multiplied by a common scaling factor so that each one of the resealed integrals is an integer number. The common scaling factor accounts for the detection sensitivity of the apparatus and for the concentration of said molecular species in the sample. The set of integer numbers thus obtained corresponds to the numbers of nuclei in the molecular species that give rise to the various signal peaks. In favorable cases of simple, non-congested spectra with small noise this procedure is successfully carried out in a straightforward fashion. Under less favorable conditions, however, calculating a set of integer rescaled intensities can result in a solution with unrealistic numbers of nuclei. For example, two signal peaks each resulting from one specific nucleus in a molecule should have the same signal integral, but finding an experimental integral ratio of 0.97/1.03 would suggest that the molecular species has 97 nuclei of the first sort and 103 nuclei of the second sort and thus would lead to an unacceptable interpretation of the NMR spectrum.
In quantitative analysis, the integral of a specific NMR signal peak of the molecular species to be determined is compared with the integral of a suitable calibration peak, which e.g. may be a synthetic NMR signal according to the ERETIC method or may be derived from a calibration substance of known concentration added to the sample. Obviously, this comparison must duly take into account the number of nuclei with identical chemical shift contributing to the specific signal peak. As in the case of qualitative analysis, any experimentally caused deviation from ideal signal integral would result in a corresponding error of the quantitative estimate. In the simple example mentioned above, depending on whether the first or the second integral is compared with the calibration integral, the result would be 0.97 or 1.03 times the true result.
In summary, the usefulness of NMR spectroscopy in qualitative and in quantitative analysis is limited by the fact that due to inevitable experimental inaccuracies and due to chemical shift degeneracy the number of nuclei of a preselected isotope in a molecular species is generally not directly available from an NMR spectrum of a sample containing said molecular species. Application of NMR spectroscopy in the fields mentioned above is also hampered by the fact that an automated determination of concentration and purity of samples is not known in the art. Automated analysis of samples with respect to number of nuclei is a prerequisite if NMR based quantitative and qualitative analysis is required at high throughput.